Protein kinase C (PKC) is a family of calcium- and phospholipid-dependent serine/threonine-specific protein kinases which play an important role in cellular growth control, regulation, and differentiation. Protein kinase C is activated by diacylglycerol (DAG), a neutral lipid, and when activated will transfer the g-phosphate of MgATP to a serine or threonine residue on a substrate protein. The mechanisms of protein kinase C action have been described in U.S. Pat. No. 4,816,450 issued Mar. 28, 1989 to Bell et al., which is incorporated herein by reference.
The activation of protein kinase C has been implicated in several human disease processes, including cancer tumors, inflammation and reperfusion injury. Accordingly, protein kinase C is a target for therapeutic agents useful in treating these conditions.
Cancer is a disease characterized in part by uncontrolled cell growth. Protein kinase C is directly involved in cellular growth control and is believed to be involved in tumor formation. Protein kinase C is fundamental to the processes involved in tumorigenicity, since it is the major high-affinity receptor for endogenous cellular DAGs as well as for several classes of tumor promoters. These tumor promoters also stimulate protein kinase C catalysis. Castagna et al., (1982) J. Biol. Chem. 257:7847, reported direct activation of protein kinase C by tumor promoting phorbol esters.
Protein kinase C is the major, if not exclusive, intracellular receptor of phorbol esters, which are very potent tumor promoters. Phorbol esters and other tumor promoters bind to and activate protein kinase C. Since DAG and phorbol esters interact at the same site, DAGs have been suggested to be the "endogenous phorbol esters", analogous to the opiate receptor where the conservation of a high affinity receptor implied the existence of an endogenous analogue. DAG has been shown to increase the affinity of protein kinase C for Ca.sup.+2 and phospholipid and thus activates protein kinase C at cellular levels of these essential cofactors. Extracellular signals including hormones, growth factors, and neurotransmitters are known to stimulate phosphatidylinositol turnover resulting in the generation of IP.sub.3 and DAG.
Structures of 40 distinct oncogenes of viral and cellular origin have revealed that oncogenes encode altered forms of normal cellular proteins. Several of the gene products appear related to growth factors or other elements involved in transmembrane signalling. These oncogene products appear to function by altering the level of critical second messengers. Cells transformed with the oncogenes ras, sis, erbB, abl, and src have been shown to contain elevated levels of DAG which is then believed to activate protein kinase C. Studies on ras transformed cells have shown protein kinase C activation to be concomitant with elevation of DAG.
Phorbol esters, such as phorbol myristate acetate (PMA), have complex effects on cells including effects on membrane function, mitogenesis, differentiation, and gene expression. Synthetic DAGs mimic many of the effects of PMA in vitro and inhibitors of protein kinase C have been shown to block PMA-induced effects on cells. Thus, protein kinase C may mediate the actions of certain oncogenes, such as ras, which cause intracellular increases in DAG and concomitant increases in protein kinase C. In addition, activation of protein kinase C leads to the expression of c-myc, c-fos, c-cis, c-fms, nuclear protooncogenes which are important in cell transformation. Overexpression of protein kinase C in NIH 3T3 cells causes altered growth regulation and enhanced tumorigenicity and in rat fibroblasts leads to anchorage-independent growth in soft agar. Overexpression of protein kinase C in these cells resulted in tumor formation in animals receiving transplanted cells.
Several studies have shown increased expression of protein kinase C in certain tumor types such as breast and lung carcinomas. Activated protein kinase C has also been detected in human colon carcinomas although increased expression at the gene level was not seen. Topoisomerases are directly modulated by protein kinase C as substrates for the enzyme.
Protein kinase C inhibitors have been reported to potentiate the antitumor activity of chemotherapeutic agents such as cis-platin both in vitro and in vivo (Grunicke et al. (1989) Adv. Enzyme Regul. 28:201; and German Offenlegungsschrift DE 3827974). In addition, it has been suggested that protein kinase C would be a potential target for therapeutic design because of its central role in cell growth (Tritton, T. R. and Hickman, J. A. Cancer Cells 2:5-102 (1990).
Inflammation and reperfusion injury, particularly pertaining to cardiac injury, are common conditions for which there exists no definitive treatment despite extensive research. Appropriate treatments for these conditions are needed.
Protein kinase C inhibitors have been demonstrated to block platelet aggregation and release of neutrophil activating agents such as platelet activating factor (PAF) (Schachtele et al. (1988) Biochem Biophy. Res. Commun. 151:542; Hannun et al (1987) J. Biol. Chem. 262:13620; Yamada et al. (1988) Biochem. Pharmacol. 37:1161). Protein kinase C inhibitors have also been shown to inhibit neutrophil activation, and chemotactic migration (McIntyre et al. (1987) J. Biol Chem. 262:15730; Lambreth et al. (1988) J. Biol. Chem. 263:3818; Pittet et al. (1987) J. Biol. Chem. 262:10072; and Gaudry et al. (1988) Immunology 63:715), as well as neutrophil degranulation and release of proteolytic enzymes and reactive oxygen intermediates (Wilson et al. (1986) J. Biol. Chem. 26:12616; Fujita et al. (1986) Biochem. Pharmacol. 35:4555; Berkow et al. (1987) J. Leukoc., Biol. 41:441; Salzer et al. (1987) Biochem. Biophys. Res. Commun. 148:747; Kramer et al. (1989) J. Biol. Chem. 262:5876; and Dewald et al. (1989) Biochem. J. 264:879).
Thus, inhibitors of protein kinase C have the capability of blocking all three of the most significant mechanisms of pathogenesis associated with myocardial reperfusion injury. Protein kinase C is, accordingly, a drug target for therapeutic agents. Additionally, the inhibitory effect of protein kinase C inhibitors on keratinocytes, and on the oxidative burst in neutrophils, provides an anti-inflammatory effect.
German Offenlegungsschrift DE 3827974 A1 discloses therapeutic preparations comprising a protein kinase C inhibitor in combination with a lipid, a lipid analogue, a cytostatic agent or phospholipase inhibitor which are useful for cancer therapy.
Cheng, C.C., (1974) J. Pharm. Sci. 63(2)307-310, disclose quinone imines as having antimalarial activity.